The integrins are heterodimeric surface molecules comprised of an .alpha. and a .beta. subunit in non-covalent association. All integrins are transmembrane proteins with counter-receptor binding activity localized in the extracellular domain. Integrins also possess relatively short cytoplasmic regions which participate in transmembrane signaling events. Integrins are capable of interacting with other cell-bound counter-receptors and components of the extracellular matrix, as well as soluble factors. Binding of extracellular ligands leads to crosslinking and localized clustering of integrins [Miyamoto, et al., Science, 267:833(1995)] and formation of focal adhesions wherein the clustered integrin cytoplasmic domains associate with cytoskeletal components including, for example, actin filaments [Pavalko and Otey, Proc. Soc. Exp. Biol. Med., 205: 32767 (1994) and Gumbiner, Neuron., 11: 551 (1993)]. While most investigations into integrin physiological activity have focused on identifying specific excellular counter-receptors, less is known about the specific interactions of integrins with cytoplasmic components. Mutation studies, however, have indicated that the cytoplasmic sequences are required for integrin association with focal contacts [LaFlamme, et al., J. Cell. Biol., 117: 437 (1992)].
While numerous integrins have been identified, certain subsets are unique to leukocytes, with each member of the subset having characteristic cell-specific expression and counter-receptor binding properties. Of leukocyte-specific integrins, at least three .beta..sub.2 integrins are known, each comprised of a unique .alpha. subunit in association with a .beta..sub.2 subunit (designated CD18) [Kishimoto, et al., Cell, 48:681-690 (1987)]. For a review of the state of the art with regard to .beta..sub.2 integrins, see Springer, Cell, 76: 301-314 (1994). CD11a/CD18, also known as .alpha..sub.1.beta..sub.2 or LGA-1, is expressed on all leukocytes and has been shown to bind to ICAM-1, ICAM-2and ICAM-3. CD11b/CD18, also know as .alpha..sub.M.beta..sub.2 or Mac-1, is expressed on polymorphonu-clear neutrophils, monocytes and eosinophils and have been shown to bind to ICAM-1, complement factor iC3b, factor X, and fibrinogen. CD11c/CD18, also known as .alpha..sub.X.beta..sub.2 or p150/95, is expressed on monocytes, polymorphonu-clear neutrophils and eosinophils and has been shown to bind to complement factor iC3c and fibrinogen. In addition, a fourth human .beta..sub.2 integrin, designated .alpha..sub.d.beta..sub.2, has recently been identified [Van der Vieren, et al., Immunity, 3: 683:690 (1995)].
The .beta..sub.7 integrin subunit, in association with an .alpha..sub.4 subunit is expressed predominately on leukocytes. The cell surface heterodimer recognizes a gut homing counterreceptor designated the mucosa addressin cell adhesion molecule (MadCAM) and appears to play a pivotal role in lymphocyte binding to intestinal tissue [Berlin, et al., Cell 74:185-195 (1993)]. .alpha..sub.4.beta..sub.7 has also been shown to bind to VCAM [Chan et al., J. Biol. Chem. 267:8366-8370 (1992)]. In a manner similar to action previously associated only with selectin surface molecules, .alpha..sub.4 .beta..sub.7 has been shown to participate in selectin-independent lymphocytes attachment to inflamed venules under flow conditions [Berlin, et al., Cell 80:413-422 (1995). Animal models using antibodies immunospecific for the .alpha..sub.4 subunit have suggested that .alpha..sub.4.beta..sub.7, or .alpha..sub.4 in associated with the .beta..sub.1 integrin subunit, may play a role in numerous disease states, including, for example, experimental allergic encephalomyelitis [Yednock, et al., Nature 356:63-66 (1992); Baron, et al., J. Exp. Med. 177:57-68 (1993)]; contact hypersensitivity [Ferguson and Kupper, J. Immunol. 150:1172-1182 (1993); Chisholm, et al., Eur. J. Immunol. 23:682-688 (1993)]; and non-obese diabetes [Yang, et al., Proc. Natl. Acad. Sci. (USA) 90:10494-10498 (1993); Burkly, et al., Diabetes 43:529-534 (1994); Baron, et al., J. Clin. Invest. 93:1700-1708 (1994)].
The integrins have been shown to be one of the major types of proteins involved in trafficking of leukocytes throughout the body. Leukocytes constantly recirculate between lymphoid organs and other tissues by passing from lymph and blood through the endothelial cell layer of the vasculature and penetrating the underlying tissue. One component of inflammatory and autoimmune diseases is the influx of leukocytes to inflammatory sites. Resolution of inflammation at such sites can be accomplished with monoclonal antibodies immunospecific for integrins which block or inhibit leukocyte function. For review, see Lobb and Hemler, J. Clin. Invest. 94:1722-1728 (1994). Modulation of integrin function is one way in which resolution of inflammation may be effected, but little is known about the specific cytoplasmic components of integrin regulation and signaling.
Liu and Pohajdak, Biochimica et Biosphysica Acta, 1132: 75-58 (1992) describes a human cDNA clone named B2-1 which encodes a protein having domains homologous to the yeast SEC7 protein, squid kinesin, pleckstrin, and dynamin. The article states that since B2-1 is homologous to only a small portion of the yeast SEC7 protein it has not been unequivocally proven that B2-1 is the human homologue of the yeast protein, which is unlikely given dissimilarities outside the SEC7 motif. The article postulates that B2-1 may be involved in the re-orientation of the golgi/secretory granules of NK cells toward target cells during cytolysis. Schiller and Kolanus ["A dominant negative effect of the human Sec7 PH domain on .beta..sub.2 integrin mediated binding to ICAM-1," 3rd Adhesion Meeting of the German Immunology Association, Regensburg, Germany, Mar. 14-15, 1995] describe a human protein having similar motifs which interacts with the cytoplasmic domain of .beta..sub.2 integrins and report that overexpression of the PH domain of the protein results in a strong dominant negative effect on the activation dependent activity of .beta..sub.2 integrins in Jurkat cells. However, Schiller and Kolanus do not show: (i) the existence of integrin regulatory proteins (IRPs) which preferentially interact with and/or modulate integrins activity. (ii) IRPs which, when co-transfected with integrin-encoding DNA in COS cells, regulate de novo expression of the co-transfected integrin, or (iii) IRPs which modulate attachment and rolling of JY cells under flow conditions.
Thus there exists a need in the art to identify molecules which bind to and/or modulate the binding and/or signaling activities of the integrins and to develop methods by which these molecules can be identified. The methods, and the molecules thereby identified, will provide practical means for therapeutic intervention in integrin-mediated immune and inflammatory responses.